The field of the invention is inhibition of complement activation.
The complement system plays a fundamental role in both the innate and acquired immune responses. As such, it also participates in the majority of diseases characterized by acute and/or chronic inflammation. For example, a critical role of the complement system has been demonstrated in rheumatoid arthritis, post-myocardial infarction reperfusion injury, post-bowel ischemia reperfusion injury, and systemic lupus erythematosus. These specific disorders are simply representative of most inflammatory states in which similar or identical molecular pathways result in complement activation and concomitant tissue injury.
Hyperacute rejection of xenografts has also been shown to result from activation of the human complement system. The utilization of organs obtained from nonhuman donors is an appealing solution to the increasing shortage of organs available for clinical transplantation. Although xenotransplantation using organs obtained from primate donors has been performed with limited clinical success, the use of distantly related species, such as pigs or sheep, avoids ethical dilemmas, potential virus transmission, and limited availability associated with the use of primates as xenograft donors. However, the use of organs from distantly related species for xenotransplantation is impractical due to hyperacute rejection (hyperacute rejection), a process that leads to irreversible xenograft damage and organ loss within minutes to hours. In xenotransplantation of vascularized tissues, hyperacute rejection is thought to be mediated by the binding of naturally occurring recipient antibodies to the endothelium of the xenograft.
The fundamental molecular basis for hyperacute rejection is thought to be activation of the classical pathway of the human complement system by human antibodies directed to immunologically foreign epitopes present on donor endothelial cells. In pig to primate xenotransplantation, it has been demonstrated that primate antibodies are primarily directed to a post-translational modification of pig membrane proteins, which modification does not occur in human cells. Specifically, foreign epitopes are generated by oligosaccharide moieties containing galactose (xcex11-3) galactose, the result of a swine enzyme that is not present in human cells. In some combinations of discordant species, activation of the alternative pathway of complement also participates in hyperacute rejection. Activation of either the classical pathway or the alternative pathway of complement leads to endothelial cell activation, thrombosis, intravascular coagulation, edema, and eventual loss of function and rejection of the transplanted organ.
The fundamental role of the complement system during hyperacute rejection has led to investigations of the potential to prevent hyperacute rejection through the use of recombinant inhibitors of the human complement system. Initial studies focused upon the use of naturally occurring endogenous human complement regulatory proteins.
Human cells and tissues are protected from inadvertent complement-mediated injury by a diverse and apparently redundant family of regulatory molecules, most of which belong to the regulators of complement activation (RCA) family. One primary function of these molecules is to inhibit formation and accumulation of C3b, which is a product of C3 cleavage by either the classical (C4b2a) or the alternative (C3bBb) pathway convertase. In general, RCA proteins can be viewed as either function-specific or pathway-specific. For example, proteins such as decay accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) are capable of regulating both the alternative and classical pathways, yet each has a limited functional role by performing either decay or cofactor function, respectively. DAF acts to dissociate the components within each of the bimolecular enzymes, whereas MCP acts as a cofactor for the serine protease factor I that cleaves C3b to form C3bi which is incapable of further participation in the complement cascade. In contrast, Factor H and C4b binding protein function specifically within the alternative pathway and classical pathway, respectively, yet each protein provides both MCP- and DAF-like control in this regard. Complement receptor type 1 (CR1) is the most versatile human complement inhibitor; it serves to regulate both the alternative pathway and classical pathway having decay and cofactor activities, and also provides a clearance function. for C3- or C4-bearing complexes which, following inactivation, it retains and transports to the reticuloendothelial system for degradation.
The functional domains of CRI, DAF, MCP, and all other RCA family members consist of repeating modules termed short consensus repeats (SCRs). Each SCR contains approximately 60 amino acids that form hypervariable domains as well as highly conserved regions. Electron microscopic studies of CRI and other family members have demonstrated that the multiple SCRs within an individual protein are tandemly arranged end to end, with each SCR representing a discrete structural unit. Each RCA family member is composed of multiple SCRs ranging from 4 SCRS in the case of DAF and MCP, to 30 SCRs in the case of the most common allotype of CR1. Amino acid homology between any two SCRs within the family ranges from 10% to 99%. These structural features of SCRs translate into the functionally conserved capacity among family members to bind C3b as well as the functionally diverse consequences of this interaction as described above.
It has been demonstrated that transgenic expression of human endogenous complement regulatory proteins (e.g. DAF, CD59) on xenografts may diminish or prevent hyperacute rejection. The human proteins that were initially chosen for these studies were presumably selected because they were among the first complement regulatory proteins to be discovered, rather than for any particular function or combinations of functions. As described above, RCA proteins are classified in three ways. A given RCA protein is described in terms of its ligands, its capacity to provide cofactor and/or decay accelerating activity, and its capacity to inhibit the alternative pathway, the classical pathway or both (Table 1). All of the RCA proteins described in Table 1 demonstrate some degree of functional versatility by inhibiting complement activation at more than one point in the cascade. Similarly, CR1 is considered the most versatile inhibitor.
Recombinant versions of regulators of complement activation have been reported. Hebel et al. (WO 91/16437, Oct. 31, 1991) describe soluble peptide analogs containing binding sites for complement. Kotwal et al. (1990, Science 250:827-830) describe a gene encoding the anti-complement protein, vaccinia virus complement control protein (VCP) (U.S. Pat. No. 5,187,268, Feb. 16, 1993), a 35 kD protein that is secreted by cells infected with vaccinia. Structurally, VCP consists entirely of four SCRs which bear 35% and 31% amino acid identity to MCP and DAF, respectively. Several studies over the past few years have demonstrated that VCP is capable of inhibiting the classical and alternative pathways through both decay and cofactor activities.
Historically, vaccinia virus was used as a vaccine to protect against infection by variola virus, the etiologic agent of smallpox. The amino acid sequence homology between most of the proteins in the two viruses is approximately 95%.
The virus that causes smallpox has been completely eradicated in that the last confirmed case of naturally occurring smallpox was reported in Somalia in October 1977. However, samples of the virus are maintained at two locations: The Centers for Disease Control in Atlanta, Ga., U.S.A. and The Russian State Research Center for Virology and Biotechnology at NPO Vector Koltsovo, Novosibirsk Region, Russia. These samples generally are not available to scientists for study. Indeed, the destruction of these samples has been the subject of serious medical and scientific debate. Consequently, little is known about the expression or function of the polypeptides encoded by the variola genome.
The variola genome is discussed in Massung et al. (1993, Nature 366:748-751). The nucleotide sequence of the genome was released into Medline (Accession No. L22579), apparently in 1995. The sequence includes an open reading frame that encodes a polypeptide with an amino acid sequence similar to the vaccinia complement inhibiting protein, VCP. The nucleotide sequences differ in twenty-six base pairs, all of which are located in the last three SCRs of VCP. However, because variola is not available for study, researchers do not know whether the variola protein has any biological activity or whether, indeed, it is produced at all. Although one might conclude that all SCR containing proteins should have some complement inhibitory activity, this is in fact not the case. For example, in addition to VCP, vaccinia virus encodes another SCR containing protein named B5R, which to date, has not been shown to exhibit any complement inhibitory activity (Herrera et al., 1998, J. Virol. 72:294-302). It was therefore necessary to produce and test the variola protein of the present invention (encoded by four SCRs) for complement inhibitory activity before concluding that it had any regulatory effect on complement.
While the complement system is essential for normal immune function in a mammal, activation of complement in certain situations may play a detrimental role in the mammal. There is a need in the art for compositions and methods which modulate complement activation, in particular, for compositions and methods which inhibit complement activation, in order that complement activation may be controlled in situations which are detrimental to a mammal. The present invention satisfies this need.
Using site-specific mutagenesis directed to a gene encoding VCP, a polynucleotide has been generated in the present invention that encodes a polypeptide which is believed to be the polypeptide encoded by an open reading frame in a published variola genome. The full-length polypeptide includes a signal sequence followed by four short consensus repeats. The short consensus repeats include four cysteine residues characteristic of the short consensus repeats of many complement binding proteins, such as CR1, CR2, C4bp, DAF, MCP and VCP. When produced as a fusion protein in which the four SCRs of the protein are attached to a chain of the Fc portion of an immunoglobulin, the protein inhibits human complement activation. The protein has greater complement-inhibiting activity than VCP.
The protein of the invention has been named xe2x80x9cSPICE,xe2x80x9d for Smallpox Inhibitor of Complement Enzymes. SPICE and proteins derived from it, are useful for the inhibition of complement activation in vitro, ex vivo and in vivo. As such, the complement inhibitors of the present invention are useful for the treatment of complement-mediated conditions. Such conditions include, but are not limited to, hyperacute rejection, as well as inflammatory diseases in which complement recruits inflammatory mediators, and reperfusion injuries.
In in vitro use, the complement inhibitors of the invention are useful for the prevention of complement-mediated damage to blood cells and for the study of complement activity.
In one aspect, the invention provides an isolated and substantially pure preparation of smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4). In another aspect, the invention provides a substantially pure preparation of a fragment of smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4), the fragment comprising at least a complement-inhibiting portion of SPICE, the portion including four short consensus repeats (SCRs) of SPICE, wherein the protein inhibits complement activation.
In another aspect, the invention provides a SPICE-related fusion protein comprising at least one polypeptide moiety attached to at least a complement-inhibiting portion of smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4), the portion including four short consensus repeats (SCRs) of SPICE, wherein the protein inhibits complement activation. In one embodiment, the polypeptide moiety increases the half-life of the protein in the human circulatory system to at least about 2 days. In another embodiment, the polypeptide moiety comprises at least a portion of an Fc molecule sufficient to bind protein A and protein G. In yet another embodiment, the polypeptide moiety comprises a signal sequence that causes secretion of the protein from a cell upon expression of the protein in the cell. In another embodiment, the polypeptide moiety comprises a transmembrane region that attaches the protein as expressed to a cell surface. In yet another embodiment, the portion of SPICE is fused to at least a transmembrane region of a regulator of complement activation selected from CR1, CR2, DAF and MCP. In yet another embodiment, the polypeptide moiety comprises at least one complement-binding SCR of a regulator of complement activation selected from the group consisting of Factor H, C4bp, CR1, CR2, DAF, MCP and VCP. In another embodiment, the portion of SPICE replaces (1) SCR1 and SCR2 of CR2 in a full-length membrane-bound or soluble CR2 protein, (2) SCRs 1-4, SCRs 8-11 or SCRs 15-18 of CR1 in a full-length membrane-bound or soluble CR1 protein or (3) at least one SCR of C4bp in a full-length C4bp polypeptide. In another embodiment, the fusion protein comprises a SPICE multimer, wherein the polypeptide moiety comprises at least one complement-inhibiting portion of SPICE. In another embodiment, the polypeptide moiety comprises a ligand that specifically binds a target receptor. In another embodiment, the polypeptide moiety comprises at least a variable region of an immunoglobulin molecule or at least a portion of an Fc molecule sufficient to bind a cell surface Fc receptor. In another embodiment, the polypeptide moiety comprises at least one constant region domain selected from CH1, CH2, and CH3, of an immunoglobulin molecule, and the protein further comprises a second polypeptide attached to the fusion protein that comprises at least a variable region of an immunoglobulin molecule, the variable region specifically binding to a ligand.
In another aspect, the invention provides a variola-regulator of complement activation (RCA) chimeric protein. The chimeric protein of the invention comprises at least one SCR from SPICE (i.e., SCR2, SCR3 or SCR4) and at least one SCR from an RCA (e.g., CR1, CR2, C4bp, DAF, MCP, Factor H and VCP). The SCRs are selected so that the chimeric protein inhibits complement activation. In one embodiment, the chimeric protein has four SCRs wherein the SCRs comprise at least one SCR from VCP and at least one SCR selected from SCR2, SCR3 or SCR4 of SPICE.
In another aspect, the invention provides a recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide that inhibits human complement activation. The polypeptide comprises at least a complement-inhibiting portion of SPICE protein (SEQ ID NO:4), the portion includes four short consensus repeats (SCRs) of SPICE. The nucleotide sequence comprises a mutated vaccinia complement control protein (VCP) sequence (SEQ ID NO: 1) encoding the portion of SPICE and substituted with codons encoding amino acid substitutions: Q96H, H117Y, S122Y, E127K, E139K, S150L, E163N, D197N, S212L, K233T, and K255Q (referring to SEQ ID NO:2). In one embodiment, the mutated VCP sequence encoding the portion of SPICE is a sequence selected from SEQ ED NO:5. In another embodiment, the recombinant polynucleotide further comprises an expression control sequence operatively linked to the nucleotide sequence. In another embodiment, the expression control sequence is operative in a mammalian cell. In another embodiment the recombinant polynucleotide is comprised within an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.
In another aspect, the invention provides a recombinant cell comprising the recombinant polynucleotide of the invention. In one embodiment, the recombinant cell is a mammalian cell.
In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a protein inhibitor of complement activation in an amount effective to inhibit human complement activation. The protein is: (a) smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4), (b) a fragment of SPICE, the fragment comprising at least a complement-inhibiting portion of SPICE, the portion including four short consensus repeats (SCRs) of SPICE, wherein the protein inhibits complement activation or (c) a SPICE-related fusion protein comprising at least one polypeptide moiety attached to at least a complement-inhibiting portion of SPICE, the portion including four SCRs of SPICE.
In another aspect, the invention provides a transgenic, non-human in mammal whose germ cells at least comprise a recombinant polynucleotide comprising an expression control sequence operative in the mammal and operatively linked to a nucleotide sequence encoding a polypeptide that inhibits human complement activation, wherein the polypeptide comprises at least a complement-inhibiting portion of smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4), the portion including four short consensus repeats (SCRs) of SPICE. In one embodiment, the non-human mammal is a non-human primate, a pig or a sheep.
In another aspect, the invention provides a graft comprising cells from a non-human mammal, the cells comprising a recombinant polynucleotide comprising an expression control sequence operative in the mammal and operatively linked to a nucleotide sequence encoding a polypeptide that inhibits human complement activation, wherein the polypeptide comprises at least a complement-inhibiting portion of smallpox inhibitor of complement enzymes (SPICE) protein (SEQ ID NO:4), the portion including four short consensus repeats (SCRs) of SPICE. In one embodiment the cells are cells from heart, lung, kidney, liver, intestine, pancreas or neural tissue.
In another aspect, the invention provides a method of making a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising at least a complement-inhibiting portion of smallpox inhibitor of complement enzyme SPICE) (SEQ ID NO:4) comprising the step of introducing into vaccinia complement control protein VCP gene sequence (SEQ ID NO:1) mutations that effect the following codon substitutions: Q96H, H117Y, S122Y, E127K, E139K, S150L, E163N, D197N, S212L, K233T, and K255Q (referring to SEQ ID NO:2). In one embodiment, the substitutions are: A288T, C349T, C365A, G379A, G415A, C449T, C450T, G487A, G489T, G589A, C635T, A698C, A763C (referring to SEQ ID NO:1).
In another aspect, the invention provides a method of inhibiting complement activation. The method comprises exposing complement to a protein inhibitor of complement activation of the invention in an amount effective to inhibit complement activation. In one embodiment, the method is for the prophylactic or therapeutic treatment of a complement-mediated condition in a human subject comprising the step of administering a pharmacologically effective amount of the protein inhibitor of complement to the subject. In another embodiment, the complement-mediated condition is hyperacute rejection, an inflammatory disorder or a post-ischemic reperfusion condition. In another embodiment, the step of administering comprises administering a vector comprising a recombinant polynucleotide encoding the protein inhibitor of complement, and the vector transfects cells of the subject and the cells express the protein inhibitor of complement.
In another aspect, the invention provides a method of inhibiting complement-mediated hyperacute rejection of a graft for transplantation. The method comprises the step of exposing the graft before implantation to a protein inhibitor of complement activation of the invention in an amount effective to inhibit complement activation. In one embodiment, the step of exposing comprises perfusing an endothelial surface of the graft with a solution comprising the complement inhibitor and a pharmaceutically acceptable carrier.
In another aspect, the invention provides a method of inhibiting hyperacute rejection of a xenograft in a human. The method comprises the step of transplanting into the human a graft comprising cells that express a protein inhibitor of complement of the invention. In one embodiment, the graft comprises organ tissue that expresses the protein as a fusion protein on the surface of endothelial cells.
In another aspect, the invention provides a method of inhibiting complement activation in blood in an extracorporeal blood loop. The method comprises the step of coating the surface of the blood loop that is exposed to blood with a protein inhibitor of complement of the invention.
In another aspect, the invention provides a blood product comprising at least a serum fraction of human blood and an amount of a protein inhibitor of complement activation of the invention.
In yet another aspect, the invention provides a method of inhibiting complement activation in a human blood product, the method comprising the step of administering to the product a protein inhibitor of complement activation of the invention.